Hi,
I am doing a pulldown in THP-1 wt lysate followed by MS-based proteomics. However I get a very low protein amount out of the cells (1 mg per 12 million cells). I use the following lysis buffer:
50 mM Tris HCl pH 7.5
150 mM NaCl
0.5% NP-40
I resuspend the pellet in the lysis buffer and rotate for 1h at 4 degrees. I do NOT use vortexing or sonication. Is this procedure sufficient to entirely lyse the cells including the nucleus? Any advice is highly appreciated.
Best,
Tim
Hello Tim,
You may try using the following recipe for non-denaturing lysis buffer, which is almost similar to your recipe except for the addition of protease and phosphatase inhibitors. Have you added these inhibitors at the time of cell lysis? Because these inhibitors are generally not stable for long periods of time.
NP-40/Triton X-100 lysis buffer:
50 mM Tris HCl, pH 8.5,
120 mM NaCl,
5mM EDTA,
1% detergent (NP40 / Triton X-100).
The concentration of salt and detergent could be altered according to your needs. Protease inhibitors should be added fresh to the lysis buffer at the time of lysis.
You may want to refer to the link below for more information.
https://www.researchgate.net/post/Can_you_suggest_me_an_extraction_protocol_of_whole_protein_or_enzyme_from_tissue_where_no_protein_denaturing_component_present_in_lysis_buffer
Good Luck!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA